The goal of the project is to use a novel approach to prevent recombinant protein misfolding and aggregation during overexpression in E. coli. The system used in this project consists of an aggregation-prone protein fused to a reporter protein (green fluorescent protein (GFP)) that reports solubility of the fusion protein in vivo. Co-expression of a third protein that binds and folds the aggregation-prone protein during protein synthesis prevents protein aggregation and results in a visible change in the signal of the reporter protein (GFP fluorescence). This system is used as a screening tool for two parallel efforts. The first is to engineer E. coli chaperones by random mutagenesis, followed by molecular evolution. The second is to construct expression libraries using yeast genomic DMA and mammalian cDNA libraries. By co-expressing libraries of chaperone mutants or eukaryotic ORFs in the E. coli cells containing the aggregation-prone protein-GFP fusion, chaperone variants, novel chaperones and folding partners that are efficient in preventing aggregation of a given recombinant protein or a family of recombinant proteins will be identified. Successful outcome of this project will help to solve one of the bottle-necks of structure genomics, lead to production of suitable amounts of proper folded proteins or protein complexes as commercial products or for structural studies, and reveal important protein-protein interactions. Structural studies of such interactions can lead to further understanding of chaperone-assisted protein folding in particular and protein folding and aggregation in general. Several neurodegenerative diseases, such as Huntington's disease, Alzheimer's disease and prion diseases, are characterized by accumulation of specific protein aggregates. Molecular chaperones have been shown to modulate solubility states of these protein aggregates. As a long-term goal, this project aims to provide insights into the mechanism of the aggregation by these disease-related proteins and the means to prevent aggregation.